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Understanding the molecular mechanisms underlying Alzheimer’s disease (AD) and finding new therapeutic targets are of utmost interest to those trying to block or at least slow down the neurodegeneration process. Both of these issues are discussed in this article that focuses on the role of a post-translational modification, the O-linked β-N-acetylglucosamine (O-GlcNAc), as a molecular mechanism preventing tau aggregation—in contrast to phosphorylation, which promotes it. During the last 10 years, O-GlcNAc has emerged as a competitor of phosphorylation for several proteins, including the microtubule-associated tau (Arnold et al., 1996). In this paper, a small-molecule inhibitor of the O-GlcNAcase (OGA), the enzyme that removes the O-GlcNAc moiety added by its opposite functional counterpart, the O-GlcNAc transferase (OGT), serves as a valuable tool to evaluate the link between phosphorylation and O-GlcNAcylation in vivo. The authors use a transgenic mouse model that overexpresses human P301L tau mutant and exhibits AD-like neurodegeneration characterized by tau hyperphosphorylation and aggregation into neurofibrillary tangles (NFTs). Treatment with OGA inhibitor leads to a significant reduction of NFTs without altering tau phosphorylation at AD-relevant sites (AT8 and pS422). In a standard in-vitro aggregation assay using anionic species as oligomerization inducers and thioflavin S fluorescence as a monitor of aggregate formation, the recombinant O-GlcNAcylated amino-terminally truncated tau (tau244-441) exhibits a slower aggregation profile than the unmodified counterpart with serine 400 (S400) playing a key role. These data indicate that O-GlcNAc modifications significantly inhibit tau aggregation in vitro, potentially by preventing the formation of soluble cytotoxic species.

Interestingly, in the P301L transgenic mouse model, reduction of NFTs concomitant with increasing level of O-GlcNAc due to OGA inhibition treatment is not accompanied by detectable changes in tau phosphorylation, at least in the proline-rich and carboxy-terminal regions, in contrast to what has been observed in a number of previous studies. In particular, S400 O-GlcNAcylation was found to both negatively regulate priming on S404 by CDK2/cyclinA3 and suppress GSK3β-mediated sequential phosphorylation of the carboxy-terminal epitope (S396/S400/S404) in vitro. Whereas implications resulting from such a disruption in the physiopathological phosphorylation process are of outmost importance in the context of AD, since GSK3β is a crucial kinase for tau phosphorylation at AD-relevant pro-directed sites, the finding that O-GlcNAc might function independently to phosphorylation suggests a new role of O-GlcNAc in AD pathogenesis. Hence, O-GlcNAc not only interferes with (hyper)phosphorylation, but also directly reduces the fibril formation on its own and, given the positive effect of increasing O-GlcNAc amounts, this study confirms the potential utility of new potent OGA inhibitors in the treatment of AD, as previously described by the authors (Yuzwa et al., 2008).

Furthermore, O-GlcNAc is here presented as a general dynamic strategy employed by the cell to protect proteins from self-assembly by stabilizing their conformation. Molecular details are needed to determine the precise function of O-GlcNAc as exemplified by S400 O-GlcNAcylation. Could a single-site glycosylation change the conformation of tau even in the presence of vicinal phosphorylations so that oligomerization is hampered? Would more O-GlcNAc modifications elicit a more potent effect? In a peptide model, no conformational change was detected upon S400 glycosylation (Smet-Nocca et al., 2011). Site-specific introduction of O-GlcNAc at the S400 position using an expressed protein ligation strategy (Broncel et al., 2012) could answer this issue at both structural and functional levels. Moreover, the use of full-length tau protein will be informative of long-range structural effects upon S400 O-GlcNAcylation, i.e., whether it disrupts the paperclip-like conformation described by Mandelkow et al. (Mandelkow et al., 2007). This transient conformation of soluble tau explains the ability of so-called discontinuous antibodies (Alz50, MC1, TG3) that detect early stages of AD to map both amino- and carboxy-termini of tau in an intramolecular manner, and also reflects the conformation adopted in paired helical filaments of tau (Bibow et al., 2011). Hence, this more compact conformation could be the signal triggering the fibrillization processes through the production of soluble cytotoxic small oligomers. In this context, one can envision O-GlcNAcylation and O-phosphorylation as opposite mechanisms that could tune tau conformation in a way that either decreases or increases the propensity to adopt a pathological conformation, respectively. As already seen with a peptide fragment from the estrogen receptor β (ERβ), both post-translational modifications might modulate ERβ activity by shifting different local structures in the intrinsically disordered amino-terminal region of the transcription factor: The phosphorylated form has a stronger propensity to adopt an extended conformation, while the O-GlcNAcylated form promotes the formation of a turn around the modification site (Chen et al., 2006). Similarly, tau O-GlcNAcylation could promote a conformational change that hinders the oligomerization process, while phosphorylation would favor a pathological conformation (Bibow et al., 2011; Sibille et al., 2011). To understand the molecular mechanisms involved in tau aggregation, as well as in regulation of tau physiological function, models such as site-specific, homogeneously modified tau proteins are required. In this context, native chemical ligation techniques could offer access to large proteins with natural protein modifications (Hackenberger et al., 2008). During the past decade, O-GlcNAc modification of tau has offered remarkable perspectives in counteracting molecular events related to AD neurodegeneration and opened new routes for AD treatments that could rival kinase inhibitors or anti-aggregative compounds. Importantly, novel methodologies are being developed to improve detection and identification of O-GlcNAc sites, which are major steps in the characterization and understanding of cellular processes (Rexach et al., 2008).